Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis

Abstract

Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO₂ retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

Keywords: Arabidopsis thaliana; Chlamydomonas reinhardtii; Rubisco; carbon concentrating mechanism (CCM); chloroplast; photosynthesis; pyrenoid; tobacco.

Figure 1

Gene expression cassettes for native and heterologous Rubisco small subunits. rbcS1A from Arabidopsis thaliana (1A_{A t}) (a), rbcS1A with α‐helices from the Chlamydomonas reinhardtii rbcS family (1A_{A t} MOD) (b), and mature rbcS2 from Chlamydomonas (S2_Cr) (c) were expressed using the rbcS1A promoter (not drawn to scale) and 35S terminator. For S2_Cr, the chloroplast transit peptide (TP) of Chlamydomonas rbcS2 (45 amino acids) was replaced with the rbcS1A TP (55 amino acids) from Arabidopsis to facilitate localisation of the mature rbcS2 to the chloroplast. (d) Alignments of the mature SSU peptides generated in this study. Numbering is relative to the Chlamydomonas rbcS2 sequence. Residues that comprise the two α‐helixes A and B are underlined, and those different from rbcS1A are in bold. For comparison with 1A_{A t} MOD, the modified spinach SSU generated by Meyer et al. (2012) is included.

Figure 2

Transcript abundances of the Rubisco gene family in rbcs mutants and transgenic lines of Arabidopsis thaliana. Abundances of rbcS1A (At1g67090), rbcS1B (At5g38430), rbcS2B (At5g38420), rbcS3B (At5g38410) and rbcL (Atcg00490) transcripts were quantified relative to wild‐type levels (set at 100) from 28‐d‐old rosettes using RT‐qPCR with gene‐specific primers (Supporting Information Table S1). For wild‐type, 1a3b and 1a2b values are the means ± SE of measurements made on three individual 28‐d‐old rosettes. For transgenic lines values are means ± SE of measurements made on nine rosettes, three from each of the three lines. Full expression data are shown in Table S2. HET, heterologous rbcS.

Figure 3

Rubisco and protein contents in rbcs mutants and transgenic lines of Arabidopsis thaliana. Rubisco (a) and total protein contents (b) are shown for 32‐d‐old plants. Rubisco content was determined via ¹⁴C‐CABP binding, and subunit ratios were estimated by immunoblotting. For wild‐type, 1a3b and 1a2b values are the means ± SE of measurements made on three individual rosettes. For transgenic lines values are means ± SE of measurements made on nine rosettes, three from each of the three lines. (c) Representative immunoblots for wild‐type plants and transgenic lines, probed with a serum containing polyclonal antibodies against Rubisco. Standard curves (0.1–2.4 μg Rubisco) are shown for wild‐type large subunit (LSU, 55 kDa) and small subunits (SSUs, 14.8 kDa), followed by protein amounts in different lines. Native LSU, SSU and heterologous SSUs (15.5 and 14 kDa, respectively) are indicated by dark grey, light grey and white arrows, respectively. Quantification of soluble protein and Rubisco is shown in Supporting Information Table S3.

Figure 4

Growth analysis of rbcs mutants and transgenic lines of Arabidopsis thaliana. (a) Representative examples of 28‐d‐old rosettes (T₃) for mutants and transgenic genotypes. (b) Rosette expansion of homozygous transgenic and 1a3b out‐segregant plants compared to that of wild‐type and 1a3b mutant plants. (c) Fresh and dry weights were compared after 28 d. For wild‐type (WT), 1a3b and 1a2b values are the means ± SE of measurements made on 10 individual rosettes. For transgenic lines values are means ± SE of measurements made on 30 rosettes, 10 from each of the three lines. See Supporting Information Table S4 for full dataset. seg, segregating T₃ wild‐type.

Figure 5

Photosynthesis response curves of rbcs mutants and transgenic lines of Arabidopsis thaliana. Measurements were made on the sixth or seventh leaf of 35‐ to 45‐d‐old nonflowering rosettes. (a) A/PAR curves show the response of CO ₂ assimilation rates to different light levels at ambient CO ₂ levels of 400 μmol mol⁻¹. (b) A/C _i curves showing the response of net CO ₂ assimilation to different sub‐stomatal concentrations of CO ₂ (C _i) under saturating light (1500 μmol photons m⁻² s⁻¹). For wild‐type, 1a3b and 1a2b values are the means ± SE of measurements made on individual leaves from four different rosettes. For transgenic lines values are means ± SE of measurements made on 12 rosettes, four from each of the three lines.

Figure 6

Nonphotochemical quenching response to light in leaves of rbcs mutants and transgenic lines of Arabidopsis thaliana. All plants were 28 d old. For wild‐type, 1a3b and 1a2b values are the means ± SE of measurements made on individual leaves from four different rosettes. For transgenic lines values are means ± SE of measurements on leaves from 12 plants, four from each of the three lines.